Nodulation Research Articles

Microbial Inoculants Drive Changes in Soil and Plant Microbiomes and Improve Plant Functions in Abandoned Mine Restoration.

The application of microbial inoculants holds promise for the sustainable restoration of abandoned mine sites by affecting soil nutrients and microbial communities. However, the responses of plant microbial communities to microbial inoculants in mine restoration remain largely unknown. To bridge this knowledge gap, we conducted a 4-year field experiment at an abandoned carbonate mine site to assess the impacts of microbial inoculants on the soil-plant microbiome. Our findings revealed that microbial inoculants significantly changed roots, fine root bacterial and fungal communities. Further, no significant correlations were observed between the soil-plant nutrient content (Z-score) and microbial alpha diversity. However, a significantly positive correlation was found between the relative abundance of the keystone ecological cluster (Module #1) and soil-plant nutrient content. The application of microbial inoculants also increased complexity, albeit decreased stability of plant microbiome networks, alongside a reduction in stochastic assembly. Conversely, they decreased the complexity but increased the stability of soil microbiome networks, accompanied by an increase in stochastic assembly. Notably, the number of specifically enriched microbiome functional traits of roots and root nodules under the microbial inoculant treatments surpassed that of the control. In summary, our findings underscored the potential of microbial inoculants to enhance soil-plant functionality at abandoned mine restoration sites.

Peptide Signals Regulate Nitrogen Deficiency Adaptation of Dicotyledonous Model Plants.

Nitrogen is a crucial macroelement essential for plant growth and development. In Arabidopsis Thaliana, classical phytohormones such as auxin and cytokinin orchestrate local and systemic signalling networks coordinate plant growth and development in response to nitrogen deficiency. Nowadays, emerging signalling pathways involving small peptides like CLAVATA3/EMBRYO SURROUNDINGR REGION (CLE) and C-TERMINALLY ENCODED PEPTIDE (CEP) and their corresponding kinase receptors, also regulate Arabidopsis' adaptation to nitrogen scarcity. Unlike Arabidopsis, which adapts to nitrogen deficiency by changing root development, legumes have the unique ability to form nitrogen-fixing root nodules through symbiotic interactions with soil rhizobia. During the symbiotic nodulation in Medicago, CLE and CEP peptides and their receptors consist of an autoregulatory network governing the number of nodules in accordance with the soil nitrogen level. Additionally, other plant peptides, such as phytosulfokine (PSK) and root meristem growth factors (RGF), have been identified as new regulators of leguminous root nodule development under nitrogen-limited condition. However, the precise mechanism by which these peptides coordinate nitrogen deficiency response and the development of nitrogen-fixing organs remains to be fully elucidated. This review summarises the adaptive strategies of dicotyledons to nitrogen deficiency, with a particular focus on the regulation of Medicago nitrogen-fixing nodule development by the peptides.

Functions of the Sinorhizobium meliloti LsrB Substrate-Binding Domain in Oxidized Glutathione Resistance, Alfalfa Nodulation Symbiosis, and Growth.

To successfully colonize legume root nodules, rhizobia must effectively evade host-generated reactive oxygen species (ROS). LsrB, a redox regulator from Sinorhizobium meliloti, is essential for symbiosis with alfalfa (Medicago sativa). The three cysteine residues in LsrB's substrate domain play distinct roles in activating downstream redox genes. The study found that LsrB's substrate-binding domain, dependent on the cysteine residue Cys146, is involved in oxidized glutathione (GSSG) resistance and alfalfa nodulation symbiosis. LsrB homologues from other rhizobia, with Cys172/Cys238 or Cys146, enhance GSSG resistance and complement lsrB mutant's symbiotic nodulation. Substituting amino acids in Azorhizobium caulinodans LsrB with Cys restores lsrB mutant phenotypes. The lsrB deletion mutant shows increased sensitivity to NCR247, suggesting an interaction with host plant-derived NCRs in alfalfa nodules. Our findings reveal that the key cysteine residue in the LsrB's substrate domain is vital for rhizobium-legume symbiosis.

Enhanced biochemical properties of soybean root nodule asparaginase through plant molecular farming compared to bacterial enzyme for cancer treatment

Asparaginase is a therapeutic enzyme used as an anticancer agent and is typically produced through microbial fermentation using organisms such as Escherichia coli and Erwinia chrysanthemi. However, this method faces challenges, including potential enzyme contamination during production, allergic reactions to the enzyme, and stability issues requiring stringent control measures. An innovative solution is the application of plant molecular farming, utilizing Rhizobium root symbiosis for asparaginase production. The objective is to optimize nodule development for asparaginase yield, characterize the enzyme's properties, and evaluate its anticancer efficacy against microbial enzyme. In our study, we established soybean root cultures and inoculated them with Bradyrhizobium japonicum to form root nodules. We evaluated eukaryotic asparaginase production at different incubation times. We purified asparaginase from the root nodule cultures and compared its physicochemical properties and anticancer activity with microbial asparaginase. Results showed that asparaginase reached maximum activity in root nodule cultures 10 days after rhizobium inoculation in the culture media. The root nodule asparaginase exhibited a high content of alpha helices and beta sheets and a low random coil. It demonstrated higher stability and activity across different pH levels and temperatures than Escherichia coli asparaginase. Additionally, root nodule asparaginase displayed better catalytic parameters and stability over time than E. coli asparaginase. Thus, root nodule asparaginase is superior to E. coli asparaginase as an anticancer agent. This ensures the root nodule asparaginase can effectively target cancer cells, enhancing the overall therapeutic outcome. This provides a renewable, cost-effective, and environmentally friendly alternative to traditional enzyme production methods.

Potential of novel Rhizobium strains as biofertilizer for improved groundnut (Arachis hypogaea L.) cultivation in Eastern India

ABSTRACT Comprehensive assessments were conducted on eight fast growing, novel Rhizobium isolates, short listed from a preliminary selection of 68 isolates obtained from root nodules of groundnut plants grown in diverse agro-climatic regions of eastern India. Additionally, two conventional rhizobia strains, viz. IGR6 and GN2, (collected from the Nodule Research Center, BCKV), were utilized for comparative analysis. Cluster analysis, based on carbon utilization and biochemical parameters, grouped all the test isolates with an average similarity level of 73%. The ability of the isolates to utilize a wide range of carbon sources, including hexoses, pentoses, and disaccharides, confirms their fast-growing nature. Further analysis through 16S rDNA extraction and nucleotide sequencing revealed that the promising fresh isolates exhibited 95–99% similarity to known Rhizobium species in public databases. When evaluated in pot culture experiments using new alluvial soil from the Gangetic Plains in eastern India, the native fresh isolate NRA1(PP355674) outperformed other isolates and conventional strains in terms of nodulation efficiency, plant dry matter production, and plant nitrogen uptake. This study highlights the significant potential of native Rhizobium isolates to surpass conventionally used strains in biological nitrogen fixation, supporting the development of sustainable, environmentally-friendly, and cost-effective agricultural practices.

Complete genome sequence of Microbacterium strain A8/3-1 isolated from the root nodule of Oxytropis tragacanthoides, growing in the Altai region.

In this article, we report the complete genome sequence of Microbacteriun strain A8/3-1 isolated from the root nodule of Oxytropis tragacanthoides Fisch. ex DC., growing in the Altai region, Russia. The sequence was obtained using Oxford Nanopore Technologies MinION.

The CEP peptide-CRA2 receptor module promotes arbuscular mycorrhizal symbiosis.

C-terminally encoded peptides (CEPs) are small secreted signaling peptides that promote nitrogen-fixing root nodulation symbiosis in legumes, depending on soil mineral nitrogen availability.1 In Medicago truncatula, their action is mediated by the leucine-rich repeat receptor-like protein kinase COMPACT ROOT ARCHITECTURE 2 (CRA2).2,3,4 Like most land plants, under inorganic phosphate limitation, M.truncatula establishes another root endosymbiotic interaction with arbuscular fungi, the arbuscular mycorrhizal symbiosis (AMS). Because this interaction is beneficial for the plant but has a high energetic cost, it is tightly controlled by host plants to limit fungal infections mainly depending on phosphate availability.5 We show in this study that the expression of a subset of CEP-encoding genes is enhanced in the low-phosphate conditions and that overexpression of the low-phosphate-induced MtCEP1 gene, previously shown to promote the nitrogen-fixing root nodulation symbiosis, enhances AMS from the initial entry point of the fungi. Conversely, a loss-of-function mutation of the CRA2 receptor required for mediating CEP peptide action2 decreases the endomycorrhizal interaction from the same initial fungal entry stage. Transcriptomic analyses revealed that the cra2 mutant is negatively affected in the regulation of key phosphate transport and response genes as well as in the biosynthesis of strigolactone hormones that are required for establishing AMS. Accordingly, strigolactone contents were drastically decreased in cra2 mutant roots. Overall, we showed that the CEP/CRA2 pathway promotes both root nodulation and AMS in legume plants, depending on soil mineral nutrient availability.

Advances and prospects in plant root nodule symbiotic nitrogen fixation

Advances and prospects in plant root nodule symbiotic nitrogen fixation

Duplication and sub-functionalization of flavonoid biosynthesis genes plays important role in Leguminosae root nodule symbiosis evolution.

Gene innovation plays an essential role in trait evolution. Rhizobial symbioses, the most important N2-fixing agent in agricultural systems that exists mainly in Leguminosae, is one of the most attractive evolution events. However, the gene innovations underlying Leguminosae root nodule symbiosis (RNS) remain largely unknown. Here, we investigated the gene gain event in Leguminosae RNS evolution through comprehensive phylogenomic analyses. We revealed that Leguminosae-gain genes were acquired by gene duplication and underwent a strong purifying selection. Kyoto Encyclopedia of Genes and Genomes analyses showed that the innovated genes were enriched in flavonoid biosynthesis pathways, particular downstream of chalcone synthase (CHS). Among them, Leguminosae-gain type Ⅱ chalcone isomerase (CHI) could be further divided into CHI1A and CHI1B clades, which resulted from the products of tandem duplication. Furthermore, the duplicated CHI genes exhibited exon-intron structural divergences evolved through exon/intron gain/loss and insertion/deletion. Knocking down CHI1B significantly reduced nodulation in Glycine max (soybean) and Medicago truncatula; whereas, knocking down its duplication gene CHI1A had no effect on nodulation. Therefore, Leguminosae-gain type Ⅱ CHI participated in RNS and the duplicated CHI1A and CHI1B genes exhibited RNS functional divergence. This study provides functional insights into Leguminosae-gain genetic innovation and sub-functionalization after gene duplication that contribute to the evolution and adaptation of RNS in Leguminosae.

Changes in root nodules dynamics under mixed cropping with varying nutrient management of berseem crop and succeeding fodder cowpea

Intercropping is a sustainable way to achieve the growing demands for food and fodder of expanding population of tropical areas. Combined application of nutrient sources along with inclusion of fodder crops in cropping system will influence soil as well as plant characteristics above ground as well as below it. These sustainable approaches may increase nutrient use efficiency by rhizosphere management at soil and plant level giving better understanding of soil plant atmosphere continuum (SPAC). The present study was conducted to evaluate the effect of nutrient management and seed ratios on dynamics of root nodules of berseem as well as succeeding crop cowpea at Agronomy Research farm, National Dairy Research Institute, Haryana, India. Intensive root studies had been done regarding root parameters in berseem as well as residual succeeding crop cowpea in this research. The study revealed that 100%RDF(Recommended dose of Fertilizer)+PGPR (Plant Growth Promoting Rhizobacteria) had given maximum number of nodules (118.60) and other traits for three cuttings in terms of nutrient management and sole berseem in terms of seed ratios number of nodules (112.92), whereas 50%RDF (Recommended dose of Fertilizer) +50% FYM (Farm Yard Manure)-(62.5) and sole berseem (54.30) at harvest had shown better results in terms of residual activity on roots nodules of succeeding crop cowpea where number of nodules (62.5) and fresh wt. of root nodules (622.1 mg) at harvest in 50%RDF + 50% FYM. This research revealed that employing organic sources of nutrients like FYM (Farm Yard Manure) and biofertilizers like PGPR (Plant Growth Promoting Rhizobacteria) along with inorganics in correct proportions enhances the root traits of leguminous fodder crops and reduce sole dependency on harmful agricultural inputs. Hence, this would fulfill the long-term nutritional requirement of crop and preserve crop status in terms of quality and quantity.

Loss of Lateral suppressor gene is associated with evolution of root nodule symbiosis in Leguminosae

BackgroundRoot nodule symbiosis (RNS) is a fascinating evolutionary event. Given that limited genes conferring the evolution of RNS in Leguminosae have been functionally validated, the genetic basis of the evolution of RNS remains largely unknown. Identifying the genes involved in the evolution of RNS will help to reveal the mystery.ResultsHere, we investigate the gene loss event during the evolution of RNS in Leguminosae through phylogenomic and synteny analyses in 48 species including 16 Leguminosae species. We reveal that loss of the Lateral suppressor gene, a member of the GRAS-domain protein family, is associated with the evolution of RNS in Leguminosae. Ectopic expression of the Lateral suppressor (Ls) gene from tomato and its homolog MONOCULM 1 (MOC1) and Os7 from rice in soybean and Medicago truncatula result in almost completely lost nodulation capability. Further investigation shows that Lateral suppressor protein, Ls, MOC1, and Os7 might function through an interaction with NODULATION SIGNALING PATHWAY 2 (NSP2) and CYCLOPS to repress the transcription of NODULE INCEPTION (NIN) to inhibit the nodulation in Leguminosae. Additionally, we find that the cathepsin H (CTSH), a conserved protein, could interact with Lateral suppressor protein, Ls, MOC1, and Os7 and affect the nodulation.ConclusionsThis study sheds light on uncovering the genetic basis of the evolution of RNS in Leguminosae and suggests that gene loss plays an essential role.

Nanoparticles Mediated Modulation of Nitrogen Fixation in Legumes: A Biochemical Perspective

This review article explores the emerging role of nanoparticles in enhancing nitrogen fixation in leguminous crops, a critical process for sustainable agriculture. Focusing on metal-based nanoparticles like iron oxide (Fe₂O₃) and zinc oxide (ZnO), as well as carbon-based nanoparticles such as carbon nanotubes (CNTs), the review discusses their effects on nitrogenase enzyme activity, root nodule formation, and nutrient uptake. Empirical studies demonstrate significant improvements in nitrogenase efficiency, nodule count, and nitrogen uptake in crops like soybean, chickpea, and peanut when treated with these nanoparticles. However, the potential risks of nanoparticle bioaccumulation, toxicity at higher concentrations, and environmental impacts are also highlighted, emphasizing the need for biodegradable nanoparticle development and regulatory guidelines. The review concludes that while nanoparticle-mediated nitrogen fixation holds promise for improving crop yields and reducing reliance on synthetic fertilizers, careful management and future research are crucial to ensuring the safe and effective integration of nanotechnology into agriculture.

The Compact Root Architecture 2 systemic pathway is required for the repression of cytokinins and miR399 accumulation in Medicago truncatula N-limited plants.

Legume plants can acquire mineral nitrogen (N) either through their roots or via a symbiotic interaction with N-fixing rhizobia bacteria housed in root nodules. To identify shoot-to-root systemic signals acting in Medicago truncatula plants at N deficit or N satiety, plants were grown in a split-root experimental design in which either high or low N was provided to half of the root system, allowing the analysis of systemic pathways independently of any local N response. Among the plant hormone families analyzed, the cytokinin trans-zeatin accumulated in plants at N satiety. Cytokinin application by petiole feeding led to inhibition of both root growth and nodulation. In addition, an exhaustive analysis of miRNAs revealed that miR2111 accumulates systemically under N deficit in both shoots and non-treated distant roots, whereas a miRNA related to inorganic phosphate (Pi) acquisition, miR399, accumulates in plants grown under N satiety. These two accumulation patterns are dependent on Compact Root Architecture 2 (CRA2), a receptor required for C-terminally Encoded Peptide (CEP) signaling. Constitutive ectopic expression of miR399 reduced nodule numbers and root biomass depending on Pi availability, suggesting that the miR399-dependent Pi-acquisition regulatory module controlled by N availability affects the development of the whole legume plant root system.

Hot Spots of Site-Specific Integration into the Sinorhizobium meliloti Chromosome.

The diversity of phage-related sequences (PRSs) and their site-specific integration into the genomes of nonpathogenic, agriculturally valuable, nitrogen-fixing root nodule bacteria, such as Sinorhizobium meliloti, were evaluated in this study. A total of 314 PRSs, ranging in size from 3.24 kb to 88.98 kb, were identified in the genomes of 27 S. meliloti strains. The amount of genetic information foreign to S. meliloti accumulated in all identified PRSs was 6.30 Mb. However, more than 53% of this information was contained in prophages (Phs) and genomic islands (GIs) integrated into genes encoding tRNAs (tRNA genes) located on the chromosomes of the rhizobial strains studied. It was found that phiLM21-like Phs were predominantly abundant in the genomes of S. meliloti strains of distant geographical origin, whereas RR1-A- and 16-3-like Phs were much less common. In addition, GIs predominantly contained fragments of phages infecting bacteria of distant taxa, while rhizobiophage-like sequences were unique. A site-specific integration analysis revealed that not all tRNA genes in S. meliloti are integration sites, but among those in which integration occurred, there were "hot spots" of integration into which either Phs or GIs were predominantly inserted. For the first time, it is shown that at these integration "hot spots", not only is the homology of attP and attB strictly preserved, but integrases in PRSs similar to those of phages infecting the Proteobacteria genera Azospirillum or Pseudomonas are also present. The data presented greatly expand the understanding of the fate of phage-related sequences in host bacterial genomes and also raise new questions about the role of phages in bacterial-phage coevolution.

Potential Nitrogen Fixing Rhizobia Isolated from Some Wild Legumes of Nagaland Based on RAPD with Nif-directed Primer and Their Biochemical Activities

Wild legumes are widely dispersed and can survive in challenging environments as bacteria dwell in their nodules and help each other. Although Nagaland is home to many wild legume varieties, research on the microbial diversity that goes along with them is still in its infancy. This work aimed to characterize several wild legume root nodules and distinguish possible rhizobial isolates using RAPD and nif-directed RPO1 primer. Nodule bacteria were isolated in Yeast extract culture media. Based on their colony morphology, 150 isolates were selected for performing RAPD with nif-directed RPO1 primer. Eighty-four isolates were bonded with RPO1 primer, and a few biochemical tests were conducted on RPO1-positive isolates. Activities that promoted plant development were also investigated for these isolates. Of all the isolates, 18 exhibited phosphate solubilization capacity, while 38 isolates were found to produce IAA. This study entails a large variety of rhizobia in the nodules, which were able to promote growth. Hence, these isolates promise to be bio-fertilizers that could improve agricultural operations.

Study of Plant Growth Promoting Abilities of Rhizobium Strains Isolated from Mungbean Root Nodules

Background: Plant Growth Promoting Rhizobacteria (PGPR) are a type of bacteria that use biofertilizers and other plant growth-promoting agents to increase plant output and growth. In the last several decades, there has been a global surge in the usage of beneficial soil microorganisms like PGPR for safe and sustainable agriculture due to the detrimental effects of artificial fertilizers on the environment and their rising prices. Plant-beneficial rhizobacteria have the potential to reduce the world's reliance on dangerous agricultural chemicals that upset agro-ecosystems. Today a large part of yield is lost due to the various stress mechanisms of plants. It could be classified into biotic and abiotic stress. These are generally a group of microorganisms that is found either in the rhizosphere or in the root nodules of plants. PGPR has beneficial impact, they colonize in the plant roots and improve crop yields and agricultural productivity and also produce some useful growth promoting growth hormones viz., IAA, Auxins, Cytokinin and Gibberellins. Methods: The bacterial isolates were screened for plant growth promoting traits such as phosphate solubilization, IAA production and siderophore production was tested qualitatively by using pikovskayas media, TSB (Tryptone Soy Broth) agar and Chrome Azurol Sulphate (CAS) blue agar respectively. Results: In present study out of the eight rhizobial isolates, five exhibited the phosphate solubilization zones, three shows negative result. However out of eight isolates five isolates produced IAA and three showed negative result. Among all isolates seven isolates were able to produce siderophore, while one showed negative result.

Effect of Seed Treatment of Boron, Cobalt and Molybdenum on Growth Parameter, Grain Yield and Seed Quality in Soybean (Glycine max L.)

This study aimed to evaluate the effect of seed treatment of boron, cobalt and molybdenum on growth parameter, grain yield and seed quality in soybean (Glycine max L.) during kharif, during July, 2022 at experimental farm, department of Soil Science and Agricultural chemistry, College of Agriculture, Badnapur. The experiment was laid out in Randomized Block Design (RBD) with 8 treatments which were replicated thrice.The treatments comprises (T1) Absolute control, (T2) RDF, (T3) RDF + Boron @ 2 gm kg-1 seed, (T4) RDF + Cobalt @ 1.5 gm kg-1 seed, (T5) RDF + Ammonium molybdate 1 gm kg-1 seed , (T6) RDF + Boron @ 2 gm kg-1 seed + Cobalt @ 1.5 gm kg-1 seed, (T7) RDF + Cobalt @ 1.5 gm kg-1 seed + Ammonium molybdate @ 1 gm kg-1 seed, (T8) RDF + Boron @ 2 gm kg-1 seed + Cobalt @ 1.5 gm kg-1 + Ammonium molybdate @ 1 gm kg-1 seed .To achieve the objectives the observations were recorded at various growth stages of soybean kharif, during 2022. The result indicates that, treatment (T8) receiving RDF + Boron @ 2 gm kg-1 seed + Cobalt @ 1.5 gm kg-1 seed + Ammonium molybdate @ 1 gm kg-1seed was found effective due to advantages effect on morpho-physiological growth, yield and yield attributing characters likeplant height, nodulation, fresh weight of nodules, no. of root nodules, root length, number of pods plant-1of soybean grain yield, Stover yield of soybean. In grain quality the results revealed that test weight, protein content percentage were significantly influenced with the application of treatment T8. On the other hand, in all the cases the lower response was found from the control treatment.

Effects of the combination of biochar and organic fertilizer on soil properties and agronomic attributes of soybean (Glycine max L.)

This research aimed to investigate the impacts of a combination of rice husk biochar and organic fertilizer on the physical and chemical properties of soil, the population of soil bacteria, the relative chlorophyll content of leaves, the development of soybean root nodules, and yield components under strongly acid soil conditions. A greenhouse and pot experiment was designed using a randomize complete block design with factorial 2 × 3 treatments and three replications. The experimental treatments comprised two rates of biochar (35 and 70 g/pot) and three rates of organic fertilizer (70, 105, and 140 g/pot). After 100 days of amendment of strongly acidic soils, the results showed that application of treatments B35F70 and B70F140 increased soil pH by 16.80% compared to the control group (CK). On the other hand, treatments B35F140 and B70F105 resulted in an increase of soil electrical conductivity by 66.67% compared to CK. In addition, after 100 days of amendment with treatments B35F105, B35F105, B35F140, B70F105, B70F70, B70F70, and B35F140, organic matter, available phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), and zinc (Zn), organic matter, available phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), and zinc (Zn), significantly increased when compared to the control group (CK). Treatment B35F140 increased relative leaf chlorophyll content and soybean seed weight per plant by 60.76% and 100.56%, respectively when compared to the CK. Furthermore, treatment B35F70 produced 125% more root nodules than CK. Moreover, each amended strongly acid soil resulted with a significant upsurge in total soil bacteria compared to the CK. Overall, statistics proved that a combination of biochar and organic fertilizer improved soil properties and soybean agronomic attributes.

Molecular diagnosis of bacteria isolated from Trifolium repens root nodules

Molecular diagnosis of bacteria isolated from Trifolium repens root nodules

Genome-wide analysis of two repeats containing MYB transcription factors in groundnut identifies drought-inducible genes involved as a negative regulator of root nodulation

The MYB gene superfamily encompasses a group of related genes found in all eukaryotes. In contrast to animals, higher plants contain large numbers of two-repeat MYB genes, which have been established in regulating crucial developmental, biotic, and abiotic stress responses that profoundly affect the plant's yield. However, a comprehensive analysis of the two-repeat MYB gene family in groundnut and its progenitors, especially the role of two-repeat MYB genes in response to drought stress, and the effect of those genes in nodulation have not been reported so far. Our recent analysis of the groundnut genome has identified 79 (Arachis duranensis), 84 (Arachis ipaensis), and 161 (Arachis hypogaea) two-repeat MYB genes, which belong to multiple distinct subgroups based on their architecture. Here, we provided a complete overview of the gene structure, protein motif organization, chromosome localization, gene duplication events, and synteny analysis to clarify evolutionary perspectives. Members of the same subgroup showed highly conserved structure and motif compositions. The whole-genome duplication and segmental duplication likely contributed to the expansion of two-repeat MYB genes in Arachis hypogaea. The upstream sequences of most genes contained phytohormone-responsive, stress-responsive, light-responsive, and plant growth-related elements. The genes not only have diverse expression profiles across different development stages but as shown in our experimental findings, most of them were induced by ABA and drought-related stress. Further gene silencing experiments demonstrated that two drought-inducible MYB genes, homologs of Arabidopsis MYB96 and MYB94, function as a negative regulator of root nodulation. The results of this study can serve as a strong foundation for further elucidation of the physiological and molecular function of two-repeat MYB genes in groundnut.